Developmental potential of small micromeres in sea urchin embryos

The large micromeres (lMics) of echinoid embryos are reported to have distinct potentials with regard to inducing endo-mesoderm and autonomous differentiation into skeletogenic cells. However, the developmental potential of small micromeres (sMics), the sibling of lMics, has not been clearly demonstrated. In this study we produced chimeric embryos from an animal cap recombined with various numbers of sMics, in order to investigate the developmental potential of sMics in the sea urchin Hemicentrotus pulcherrimus and the sand dollar Scaphechinus mirabilis. We found that sMics of H. pulcherrimus had weak potential for inducing presumptive ectoderm cells to form endo-mesoderm structures. The inducing potential of ten sMics was almost equivalent to that of one lMic. The sMics also had the potential to differentiate autonomously into skeletogenic cells. Conversely, the sMics of S. mirabilis did not show either inductive or skeletogenic differentiation potential. The sMics of both species had the potential to induce oral-aboral axis establishment. These results suggest that the potential for sMics to differentiate into skeletogenic cells and for inducing the presumptive ectoderm to differentiate into endomesoderm differs across species, while the potential of sMics to induce the oral-aboral axis is conserved among species.     

Mass isolation and culture of sea urchin micromeres

Summary A procedure is described for large-scale isolation of micromeres from 16-cell stage sea urchin embryos. One to two grams of >99% pure, viable micromeres (2.3 to 4.6 × 10⁸ cells) are routinely isolated in a single preparation. In culture, these cells uniformly proceed through their normal development, in synchrony with micromeres in whole embryos, ultimately differentiating typical larval skeletal structures. The attributes of this procedure are: (a) the very early time of isolation of the cells, directly after the division that establishes the cell line; (b) the large yield of cells; (c) the purity of the preparation of cell; and (d) their synchronous development in culture through skeletogenesis. The procedure greatly aids in making sea urchin micromeres a favorable material for molecular analysis of development. This work was supported in part by the following grants from the National Institutes of Health: Grant HL-10312 to A.H.W., Grant GM-20784 to Helen R. Whiteley, Grant ES-02190 to N. Karle Mottet, M.D., and Training Grants ES-07032 and HD-00266.     

Small micromeres contribute to the germline in the sea urchin

Many indirect developing animals create specialized multipotent cells in early development to construct the adult body and perhaps to hold the fate of the primordial germ cells. In sea urchin embryos, small micromeres formed at the fifth division appear to be such multipotent cells: they are relatively quiescent in embryos, but contribute significantly to the coelomic sacs of the larvae, from which the major tissues of the adult rudiment are derived. These cells appear to be regulated by a conserved gene set that includes the classic germline lineage genes vasa, nanos and piwi. In vivo lineage mapping of the cells awaits genetic manipulation of the lineage, but previous research has demonstrated that the germline is not specified at the fourth division because animals are fertile even when micromeres, the parent blastomeres of small micromeres, are deleted. Here, we have deleted small micromeres at the fifth division and have raised the resultant larvae to maturity. These embryos developed normally and did not overexpress Vasa, as did embryos from a micromere deletion, implying the compensatory gene regulatory network was not activated in small micromere-deleted embryos. Adults from control and micromere-deleted embryos developed gonads and visible gametes, whereas small micromere-deleted animals formed small gonads that lacked gametes. Quantitative PCR results indicate that small micromere-deleted animals produce background levels of germ cell products, but not specifically eggs or sperm. These results suggest that germline specification depends on the small micromeres, either directly as lineage products, or indirectly by signaling mechanisms emanating from the small micromeres or their descendants.     

Analysis of competence in cultured sea urchin micromeres.

Sea urchin embryo micromeres form the primary mesenchyme, the skeleton-producing cells of the embryo. Almost nothing is known about nature and timing of the embryonic cues which induce or initiate spicule formation by these cells. A related question concerns the competence of the micromeres to respond to the cues. To examine competence in this system we have exposed cultured sea urchin micromeres to an inducing medium containing horse serum for various periods of time and have identified a period when micromeres are competent to respond to serum and form spicules. This window, between 30 and 50 h after fertilization, corresponds to the time when mesenchyme cells in vivo are aggregating and beginning to form the syncytium in which the spicule will be deposited. The loss of competence after 50 h is not due to impaired cell health since protein synthesis at this time is not significantly different from controls. Likewise the accumulation of a spicule matrix mRNA (SM 50) and a cell surface glycoprotein (msp 130), both indices of micromere/mesenchyme differentiation, still occurs in cells that have lost competence to respond to serum by forming spicules. These experiments demonstrate that the acquisition and loss of competence in these cells are regulated developmental events and establish an in vitro system for the identification of the molecular basis for inductive signal recognition and signal transduction.     

Collagen metabolism and spicule formation in sea urchin micromeres

The role of collagen or collagen-like protein(s) in the in vitro formation of the sea urchin embryonic skeleton was investigated using isolated micromeres of Strongylocentrotus purpuratus. Micromeres were cultured in sea water containing 4% horse serum on tissue culture plastic or an extracellular matrix of type I collagen. The effect of proline analogs and an inhibitor of collagen hydroxylation on in vitro spicule formation in both culture systems was monitored. When micromeres are cultured in the presence of proline analogs l-azetidine-2-carboxylic acid and l-3,4-dehydroproline which disrupt collagen metabolism, spicule formation is significantly less inhibited on a collagen substratum than on plastic. Culturing micromeres on plastic in the presence of α,α′-dipyridyl, an inhibitor of collagen hydroxylation, resulted in almost complete inhibition of spicule formation. The inhibition by α,α′-dipyridyl can be overcome by culturing micromeres on collagen substratum. These results do not support the idea of collagen being the calcified organic matrix of the spicule. Rather, they suggest that micromeres synthesize a collagen-like extracellular matrix which is necessary for spicule formation. Inhibition of this activity by proline analogs or a collagen processing inhibitor can be overcome by providing the cells with a previously deposited extracellular matrix.     

Serum effects on the in vitro differentiation of sea urchin micromeres

When micromeres isolated from the 16-cell stage of Strongylocentrotus purpuratus are cultured in sea water containing 3.5% horse serum, they produce spicules at approximately the same time as in normal development. The serum requirement of the micromeres has been investigated by adding serum at varying intervals after isolation or by pulsing the cells with serum at specific times during their in vitro development. The optimum time of serum addition for spicule formation is 36 h after fertilization (AF). Further delay in the addition of serum results in a reduction in the number of spicules formed in culture and a delay in the time at which they appear. A 1-h pulse of serum at 36 h AF is sufficient to initiate a response in some of the micromere aggregates. A 12-h pulse at 36 h AF produces the maximum number of spicules per culture. The critical period for serum addition, 36-48 h AF, corresponds to the time in the normal embryo at which the syncytial primary mesenchyme ring is formed. Electron micrographs of cultured cells demonstrate that micromeres cultured without serum until 48 h AF fail to form pseudopodial extensions and remain as rosette-like clusters of cells. If serum is present, extensive pseudopodial networks form which resemble the primary ring syncytium. These results suggest that serum acts to stimulate fused pseudopodial networks in cultures of micromeres and that the resulting syncytium is necessary for spicule formation.     

Time-lapse and electron microscopic studies of sea urchin micromeres

Summary The cleavage pattern of the young sea urchin embryo was studied by means of light and electron microscopy.The micromeres, which are known to have a strong organizing effect on the embryo, were found to form a syncytium with their neighbouring micromeres and with the macromeres. The cell walls between these cells were observed to be incomplete while there were interphase nuclei with intact nuclear membranes in the micro- and the macromeres. Similar phenomena with a break down of the cell membranes were not observed between macro- and mesomeres while there were intact interphase nuclei in these cells. Micromeres implanted on macromeres or mesomeres were found to coalesce with these latter cells in the course of a few minutes. During interphase, when the nuclei of both micro- and mesomere (macromere) had intact nuclear membranes, there also was a break down of the cell walls and a syncytium was formed by the host cell and the implanted micromere (see Fig. 6).The primary mesenchyme cells, which are regarded as the descendants of the micromeres, were also studied and were likewise found to form true syncytia.The importance to embryogenesis of this unique formation of syncytia is discussed.     

The synthesis and secretion of collagen by cultured sea urchin micromeres

Circumstantial evidence in several previous studies has suggested that sea urchin embryo micromeres, the source of primary mesenchyme cells which produce the embryonic skeleton, contribute to the extracellular matrix of the embryo by synthesizing collagen. A direct test of this possibility was carried out by culturing isolated micromeres of the sea urchin Stronglyocentrotus purpuratus in artificial sea water containing 4% (v/v) horse serum. Under these conditions the micromeres divide and differentiate to produce spicules with the same timing as intact embryos. Collagen synthesis was determined by labeling cultures with [3H]proline or [35S]methionine and the medium and cell layer were assayed for collagen. The results indicate that by the second day in culture micromeres synthesize and secrete a collagenase-sensitive protein doublet with a molecular weight of about 210 kDa. Densitometry indicates a 2:1 ratio of the respective bands in the doublet which is characteristic of Type I collagen. The doublet is insensitive to digestion with pepsin. This differential sensitivity is characteristic of collagen. Over 90% of the collagen synthesized by micromeres is soluble in the seawater culture medium. On days 2-4 in culture, collagen accounts for 5% of the total protein synthesized and secreted. Additional collagenase-sensitive bands are noted at 145 and 51 kDa. The relationship of the described collagen metabolism to previously characterized collagen gene expression in sea urchin embryos is discussed.     

Limited complexity of the RNA in micromeres of sixteen-cell sea urchin embryos

The sequence complexity of sea urchin embryo micromere RNA is about 75% of that of total 16-cell embryo cytoplasmic RNA, as reported earlier by Rodgers and Gross [Rodgers, W. H., and Gross, P. R. (1978) Cell, 14, 279–288]. In contrast to the rest of the embryo, there are few, if any, complex maternal RNA species in the micromere cytoplasm which are not represented in the polysomes. The micromeres do not contain detectable quantities of high-complexity nuclear RNA, though such RNA exists in other cells of the fourth-cleavage embryo.     

Vasa protein expression is restricted to the small micromeres of the sea urchin, but is inducible in other lineages early in development

Vasa is a DEAD-box RNA helicase that functions in translational regulation of specific mRNAs. In many animals it is essential for germ line development and may have a more general stem cell role. Here we identify vasa in two sea urchin species and analyze the regulation of its expression. We find that vasa protein accumulates in only a subset of cells containing vasa mRNA. In contrast to vasa mRNA, which is present uniformly throughout all cells of the early embryo, vasa protein accumulates selectively in the 16-cell stage micromeres, and then is restricted to the small micromeres through gastrulation to larval development. Manipulating early embryonic fate specification by blastomere separations, exposure to lithium, and dominant-negative cadherin each suggest that, although vasa protein accumulation in the small micromeres is fixed, accumulation in other cells of the embryo is inducible. Indeed, we find that embryos in which micromeres are removed respond by significant up-regulation of vasa protein translation, followed by spatial restriction of the protein late in gastrulation. Overall, these results support the contention that sea urchins do not have obligate primordial germ cells determined in early development, that vasa may function in an early stem cell population of the embryo, and that vasa expression in this embryo is restricted early by translational regulation to the small micromere lineage.     

Phorbol esters alter cell fate during development of sea urchin embryos

Protein kinase C (PKC) has been implicated as important in controlling cell differentiation during embryonic development. We have examined the ability of 12-O-tetradecanoyl phorbol-13-acetate (TPA), an activator of PKC, to alter the differentiation of cells during sea urchin development. Addition of TPA to embryos for 10-15 min during early cleavage caused dramatic changes in their development during gastrulation. Using tissue-specific antibodies, we have shown that TPA causes the number of cells that differentiate as endoderm and mesoderm to increase relative to the number that differentiate as ectoderm. cDNA probes show that treatment with TPA causes an increase in accumulation of RNAs specific to endoderm and mesoderm with a concomitant decrease in RNAs specific to ectoderm. Treatment of isolated prospective ectodermal cells with TPA causes them to differentiate into endoderm and mesoderm. The critical period for TPA to alter development is during early to mid cleavage, and treatment of embryos with TPA after that time has little effect. These results indicate that PKC may play a key role in determining the fate of cells during sea urchin development.     

Determination of cell fate in sea urchin embryos

Classical embryological studies have provided a great deal of information on the autonomy and stability of cell fate determination in early sea urchin embryos. However, these studies were limited by the tools available at the time, and the interpretation of the results of these experiments was limited by the lack of information available at the molecular level. Recent studies which have re-examined classical experiments at the molecular level have provided important new insights into the mechanism of determination in sea urchins, and require us to re-evaluate some long standing theories on the process of differentiation.     

Nanos functions to maintain the fate of the small micromere lineage in the sea urchin embryo

The translational regulator nanos is required for the survival and maintenance of primordial germ cells during embryogenesis. Three nanos homologs are present in the genome of the sea urchin Strongylocentrotus purpuratus, all of which are expressed with different timing in the small micromere lineage. This lineage is set-aside during embryogenesis and contributes to constructing the adult rudiment. Small micromeres lacking Sp-nanos1 and Sp-nanos2 undergo an extra division and are not incorporated into the coelomic pouches. Further, these cells do not accumulate Vasa protein even though they retain vasa mRNA. Larvae that develop from Sp-nanos1 and 2 knockdown embryos initially appear normal, but do not develop adult rudiments; although they are capable of eating, over time they fail to grow and eventually die. We conclude that the acquisition and maintenance of multipotency in the small micromere lineage requires nanos, which may function in part by repressing the cell cycle and regulating other multipotency factors such as vasa. This work, in combination with other recent results in Ilyanassa and Platynereis dumerilii, suggests the presence of a conserved molecular program underlying both primordial germ cell and multipotent cell specification and maintenance.     

Changes in cell surface charges during differentiation of isolated micromeres and mesomeres from sea urchin embryos

Changes in the negative surface charge were observed by cell electrophoresis during the differentiation of micromeres and mesomeres isolated from 16-cell-stage sea urchin embryos. Micromeres and mesomeres were separated by a sucrose density gradient column and were cultured in normal seawater. An isolated micromere developed to a cell aggregate, and, at the mesenchyme-blastula stage of control, the aggregate began to scatter into single cells. These processes are quite similar to those of the primary mesenchyme cells in situ. An isolated mesomere, on the other hand, developed into an ectodermal vesicle. At desired stages of development, the cell aggregates which derived from single blastomeres were dissociated into single cells, and their electrophoretic mobilities were measured. It was found that the electrophoretic mobility of the micromere- and mesomere-derived cells concomitantly increased from the early blastula stage up to the early mesenchyme stage. In contrast with the mesomere-derived cells, however, the micromere-derived cells showed another increase in electrophoretic mobility when the cells began to migrate as primary mesenchyme cells. These results show that a correlation exists between the increase in cell surface negative charge and the migration of the primary mesenchyme cells.